Crystal form of heterocyclic compound and pharmaceutical composition thereof

Abstract

The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a crystal form of a heterocyclic compound and a pharmaceutical composition thereof. Specifically, the present invention relates to a crystal form of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-formamido)pyridine-2-formamide and a pharmaceutical composition thereof. The crystal form of the present invention has good crystallinity, excellent properties in terms of chemical stability and crystal stability, high process safety, and good drug efficacy in a variety of animal models.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and relates to a crystal form of a heterocyclic compound and a pharmaceutical composition thereof. Specifically, the present invention relates to a crystal form of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide and a pharmaceutical composition thereof. Background Art

[0002] Opioids are alkaloids and their analogs that can interact with central specific receptors and are often used to treat pain. Repeated use of opioids will cause the body to tolerate and become addicted. While opioids exert their efficacy, they are usually accompanied by many problems such as safety, tolerance, and addiction. Therefore, the research and development of non-opioid acute pain drugs has a huge potential market space.

[0003] In recent years, researchers have found that NaV1.8 is an important sodium ion channel for pathological and pain-related diseases, and plays a key role in pain signal transmission in the peripheral nervous system, chronic neuropathic pain, and chronic inflammatory pain. This mode of action is completely different from opioids. It does not act on the brain, but directly blocks pain in the peripheral nervous system. It is relatively safe, non-addictive, and does not have the gastrointestinal side effects and cardiovascular and cerebrovascular side effects of nonsteroidal anti-inflammatory drugs. It can be used in combination with other analgesics to enhance efficacy and reduce side effects.

[0004] Researchers have discovered a series of NaV1.8 blockers, including the small molecule NaV1.8 blocker VX-548 from Vertex, USA. Vertex, USA, also made structural modifications based on VX548, such as WO2022256676. We have also discovered a class of NaV1.8 blockers, which are recorded in Chinese patent application CN202311551257.1.

[0005] The regulatory mechanism of Nav1.8 expression is still unclear, but further research is needed to meet clinical needs such as high selectivity, strong efficacy, low side effects and non-addiction.

[0006] Through extensive research, the present application has found that the compounds of the present invention have properties that are more suitable for medical and pharmaceutical uses under certain conditions, such as better solubility, bioavailability, physical and / or chemical stability, purity and impurity characteristics, filtration properties, drying properties, hygroscopicity or easier processing. In addition to being easy to industrialize, the compounds of the present invention exhibit excellent efficacy and good safety in a variety of models. Summary of the invention

[0007] The first aspect of the present invention provides a crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide.

[0008] The second aspect of the present invention provides a pharmaceutical composition comprising the crystalline form A and one or more pharmaceutically acceptable carriers.

[0009] The third aspect of the present invention provides a preparation comprising the crystalline form A and one or more pharmaceutically acceptable carriers.

[0010] The fourth aspect of the present invention provides a medicine kit, which comprises the crystalline form A or the pharmaceutical composition or preparation, optionally further comprises one or more other therapeutic agents, and a drug instruction sheet.

[0011] The fifth aspect of the present invention provides the use of the above-mentioned crystal form A, pharmaceutical composition, preparation or kit in the preparation of a drug for preventing and / or treating a NaV1.8-related disease. Preferably, the NaV1.8-related disease is pain.

[0012] The sixth aspect of the present invention provides the above-mentioned crystal form A, pharmaceutical composition, preparation or medicine kit for preventing and / or treating NaV1.8-related diseases. Preferably, the NaV1.8-related disease is pain.

[0013] The seventh aspect of the present invention provides a method for preventing and / or treating NaV1.8-related diseases, comprising administering to an individual a therapeutically effective amount of the above-mentioned crystal form A, pharmaceutical composition, preparation or kit; preferably, the NaV1.8-related disease is pain.

[0014] The present invention has the following beneficial effects: the compound 1 crystal form A of the present invention has high crystallinity, high purity, uniform particle size distribution, no hygroscopicity, excellent thermal stability and high process safety, simple operation, and is suitable for industrial production. The compound of the present invention has good solubility and bioavailability. The compound of the present invention also shows excellent efficacy and good safety in multiple models. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the XRPD pattern of Compound 1 Form A. DETAILED DESCRIPTION

[0016] The present invention is further described below through examples. The examples of the present invention are only used to illustrate the technical solution of the present invention and are not used to limit the scope of the present invention. Those skilled in the art may make some non-essential improvements and adjustments, which still fall within the protection scope of the present invention.

[0017] definition

[0018] Unless otherwise stated, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. If there is a conflict, the definition herein shall prevail. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0019] The term "Compound 1" as used herein refers to the compound 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide.

[0020] The term "about" when used with a numerical variable generally refers to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within a range of ±20%, ±10%, ±5% or ±2% of the stated value.

[0021] The term "comprising" or its synonymous similar expressions "including", "containing" and "having" are open-ended and do not exclude additional unlisted elements, steps or components. The expression "consisting of..." excludes any elements, steps or components not specified.

[0022] The term "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, plus the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the term "comprising" and similar terms encompass the terms "consisting essentially of" and "consisting of."

[0023] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring of said event or circumstance.

[0024] Unless otherwise indicated, percentages, parts, etc. herein are all by weight.

[0025] As used herein, the term "crystalline form" or "crystal" refers to any solid material that exhibits a three-dimensional ordering, in contrast to amorphous solid material, which produces a characteristic XRPD pattern with well-defined peaks.

[0026] As used herein, the term "seed crystals" refers to an additive in a crystallization process that can form crystal nuclei to accelerate or promote the growth of crystals of the same enantiomer as the crystal form or stereoconfiguration.

[0027] As used herein, the term "X-ray powder diffraction pattern" or "XRPD pattern" refers to an experimentally observed diffraction pattern or a parameter, data or value derived therefrom. An XRPD pattern is typically characterized by peak positions (abscissa) and / or peak intensities (ordinate).

[0028] As used herein, the term "diffraction angle" or "2θ" refers to the peak position expressed in degrees (°) based on the setting in the X-ray diffraction experiment, and is usually the unit of the abscissa in the diffraction pattern. If the reflection is diffracted when the incident beam forms an angle θ with a certain lattice plane, the experimental setting needs to record the reflected beam at an angle of 2θ. It should be understood that the specific 2θ value of a specific crystalline form mentioned in this article is intended to represent the 2θ value (expressed in degrees) measured using the X-ray diffraction experimental conditions described herein. For example, as described herein, Cu-Kα (Kα1 (Å): 1.5406) ​​monochromatic radiation is used. The XRPD spectrum herein is preferably collected on a PANalytacal X'Pert3 Powder X-ray powder diffraction analyzer, and the transmission mode is preferably collected on a PANalytacal X'Pert3 Powder X-ray powder diffraction analyzer.

[0029] As used herein, the term "substantially the same" or "substantially as shown in Figure X" for X-ray diffraction peaks means that representative peak position and intensity variations are taken into account. For example, those skilled in the art will understand that peak position (2θ) will show some variation, typically as much as 0.1 to 0.2 degrees, and that the instrument used to measure diffraction will also cause some variation. In addition, those skilled in the art will understand that relative peak intensities will vary due to differences between instruments, as well as the degree of crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art.

[0030] Similarly, as used herein, "substantially as shown in Figure X" for DSC spectra and TGA spectra is also intended to cover variations associated with these analytical techniques known to those skilled in the art. For example, for well-defined peaks in DSC spectra, there will typically be variations of up to ±5°C, and even greater (e.g., up to ±10°C) for broad peaks.

[0031] As used herein, the term "room temperature" refers to 20°C ± 5°C.

[0032] As used herein, the term "prevention" includes inhibiting and delaying the onset of a disease, and includes not only prevention before the development of a disease but also prevention of recurrence of a disease after treatment.

[0033] As used herein, the term "treat" refers to reversing, alleviating or eliminating the targeted disease or condition. If a subject receives a therapeutic amount of a salt of a compound of the present invention or a compound in a crystalline form thereof or a pharmaceutical composition of the present invention, and at least one indicator and symptom of the subject shows observable and / or detectable relief and / or improvement, it indicates that the subject has been successfully "treated". It is understood that treatment includes not only complete treatment, but also includes not achieving complete treatment but achieving some biological or medically relevant results. Specifically, "treat" means that a salt of a compound of the present invention or a compound in a crystalline form thereof or a pharmaceutical composition of the present invention can achieve at least one of the following effects: (1) inhibiting the disease in an animal that is experiencing or showing disease pathology or symptoms (i.e., preventing further development of the pathology and / or symptoms); (2) ameliorating the disease in an animal that is experiencing or showing disease pathology or symptoms (i.e., reversing the pathology and / or symptoms). DETAILED DESCRIPTION OF THE INVENTION

[0035] Compound 1 Form A

[0036] The present invention provides a crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide, wherein the XRPD spectrum of the crystalline form A includes a wavelength of 12.8±0.2 o , 14.5±0.2 o and / or 21.9 ± 0.2 o Characteristic peak at the diffraction angle (2θ) of .

[0037] In some embodiments, the diffraction angle (2θ) in the XRPD pattern of Form A is 14.5°±0.2 o The diffraction peak intensity is 100%, and the diffraction angle (2θ) is 12.8°±0.2 o The diffraction peak intensity is ≥70%.

[0038] In some embodiments, the XRPD pattern of Form A includes o , 13.2±0.2 o , 14.5±0.2 o , 19.3±0.2 o and / or 21.9 ± 0.2 o Characteristic peak at the diffraction angle (2θ) of .

[0039] In some embodiments, the XRPD pattern of Form A includes o , 13.2±0.2 o , 14.5±0.2o , 17.8±0.2 o , 19.3±0.2 o and / or 21.9 ± 0.2 o Characteristic peak at the diffraction angle (2θ) of .

[0040] In some embodiments, the XRPD pattern of Form A includes o , 13.2±0.2 o , 14.5±0.2 o , 17.8±0.2 o , 19.3±0.2 o , 20.6±0.2 o , 21.9 ±0.2 o , 25.0±0.2 o , 25.6±0.2 o and / or 27.0±0.2 o Characteristic peak at the diffraction angle (2θ) of .

[0041] In some embodiments, the XRPD pattern of Form A includes o , 13.2±0.2 o , 14.5±0.2 o , 16.0±0.2 o , 17.8±0.2 o , 18.6±0.2 o , 19.3±0.2 o , 19.6±0.2 o , 20.6±0.2 o , 21.4 ±0.2 o , 21.9 ±0.2 o , 23.6±0.2 o , 24.3±0.2 o , 25.0±0.2 o , 25.6±0.2 o , 26.0±0.2 o , 27.0±0.2 o , 27.9±0.2 o , 28.5±0.2 o , 29.3±0.2 o , 36.3±0.2 o Characteristic peak at the diffraction angle (2θ) of .

[0042] In some embodiments, the XRPD pattern of Form A includes Figure 1 The peaks at substantially the same diffraction angles (2θ) are shown.

[0043] In some embodiments, the XRPD pattern of Form A is substantially as follows Figure 1 shown.

[0044] In some embodiments, the XRPD pattern of Form A is as follows Figure 1 shown.

[0045] Pharmaceutical compositions, preparations and kits

[0046] The pharmaceutical composition provided by the present invention comprises the crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide and one or more pharmaceutically acceptable carriers.

[0047] In some embodiments, the pharmaceutical composition comprises 0.01-10000 mg of the crystalline form A, and one or more pharmaceutically acceptable carriers.

[0048] In some embodiments, the pharmaceutical composition comprises 0.03-8000 mg of the crystalline form A, for example 0.05-8000 mg, 0.1-8000 mg, 10-8000 mg, 10-1000 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg g, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg , 1500mg, 2000mg, 2500mg, 3000mg, 3500mg, 4000mg, 4500mg, 5000mg, 5500mg, 6000mg, 6500mg, 7000mg, 7500mg, 8000mg.

[0049] The preparation provided by the present invention comprises the crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide and one or more pharmaceutically acceptable carriers.

[0050] In some embodiments, the preparation comprises 0.01-10000 mg of the crystalline form A, and one or more pharmaceutically acceptable carriers.

[0051] The medicine kit provided by the present invention comprises the crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide or the pharmaceutical composition or the preparation, and optionally further comprises one or more other therapeutic agents.

[0052] Treatments and uses

[0053] The present invention provides a crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide, a pharmaceutical composition, a preparation or a medicine kit for preparing a drug for preventing and / or treating NaV1.8-related diseases. Preferably, the NaV1.8-related disease is pain.

[0054] The present invention provides a crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide, a pharmaceutical composition, a preparation or a medicine kit for preventing and / or treating NaV1.8-related diseases. Preferably, the NaV1.8-related disease is pain.

[0055] The present invention provides a method for preventing and / or treating NaV1.8-related diseases, comprising administering to an individual a therapeutically effective amount of crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide; preferably, the NaV1.8-related disease is pain.

[0056] In some embodiments, the pain is selected from chronic pain, acute pain, inflammatory pain, cancer pain, ICU analgesia, fracture or postoperative pain (e.g., bunionectomy pain, hernia repair pain, and abdominoplasty pain), neuropathic pain (e.g., peripheral neuropathy, postherpetic neuralgia, peripheral neuropathy, small fiber neuropathy pain, trigeminal neuralgia, idiopathic small fiber neuralgia, or diabetic neuropathy), visceral pain (e.g., intestinal pain), musculoskeletal pain, primary pain, idiopathic pain, osteoarthritis pain, gouty arthritis pain, rheumatic or rheumatoid arthritis pain, dental pain, joint pain, labor pain, fibromyalgia, chronic low back pain, bladder pain syndrome, and sciatica.

[0057] In some embodiments of the present invention, the pain is selected from postoperative pain, neuropathic pain (e.g., postherpetic neuralgia, small fiber neuropathy pain, diabetic neuropathy), osteoarthritis pain, bladder pain syndrome, and cancer pain.

[0058] Preparation method of crystal form

[0059] In some embodiments, the present invention provides a method for preparing Compound 1 Form A, which comprises mixing the product obtained by chiral separation of compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylic acid methyl ester with a good solvent, and then adding a poor solvent to separate the obtained solid.

[0060] In some embodiments, the preparation method of Compound 1 Form A specifically includes the following steps: the product obtained by chiral separation of compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylic acid methyl ester is mixed with a good solvent, then cooled to 50-60°C, a poor solvent is added, and then cooled to 0-30°C to separate the obtained solid.

[0061] In some embodiments, the preparation method of Compound 1 Form A specifically includes the following steps: the product obtained by chiral separation of compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylic acid methyl ester is mixed with methanol and heated to dissolve, then cooled to 50-60°C, water is added, and then cooled to 0-30°C, crystallized, and the obtained solid is separated.

[0062] In some embodiments, in the preparation method of Compound 1 Form A, the mass volume ratio of the product obtained by chiral separation of compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2carboxylic acid methyl ester to the good solvent is 1:2-30 in g / ml, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30.

[0063] In some embodiments, in the preparation method of Compound 1 Crystalline Form A, the mass volume ratio of the product obtained by chiral separation of compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2carboxylic acid methyl ester to the poor solvent is 1:5-30 in g / ml, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30.

[0064] In some embodiments, the chiral separation conditions are chromatographic column AD-H, column temperature 20-30° C., and a mobile phase selected from a mixed solution of n-hexane-anhydrous ethanol-isopropanol-diethylamine.

[0065] In some embodiments, the flow rate for the chiral separation is selected from 1-10 mL / min.

[0066] In some embodiments, the volume ratio of n-hexane-anhydrous ethanol-isopropanol-diethylamine in the mobile phase for chiral separation is selected from (70-90): (10-20): (1-10): (0.01-1).

[0067] In some embodiments, the volume ratio of n-hexane-anhydrous ethanol-isopropanol-diethylamine in the mobile phase for chiral separation is 80:16:4:0.05.

[0068] In some embodiments, the chiral separation product is a component with a retention time of about 5.3 min, such as 5.2 min, 5.3 min, and 5.4 min.

[0069] In some embodiments, the Compound 1 Form A provided by the present invention can be prepared using the preparation method described above.

[0070] The crystalline forms or amorphous forms prepared in the examples were characterized by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), polarizing light microscopy (PLM), dynamic water sorption (DVS) and X-ray single crystal diffractometer.

[0071] (1) X-ray powder diffraction (XRPD)

[0072] The X`Pert3 Powder powder diffractometer was used to collect the XRPD patterns of the crystalline or amorphous forms. The instrument was irradiated with Cu palladium and detected using Absolute scan at room temperature. The detection range was 3.5° to 40°, the step length was 0.013°, the dwell time was 50s, and the scan was 1 time.

[0073] (2) Differential Scanning Calorimetry (DSC)

[0074] The DSC spectrum of the crystal form was collected using a TA DSC 2500 differential scanning calorimeter. The test temperature range was 35°C to 250°C, and the heating rate was 10°C / min.

[0075] (3) Thermogravimetric analysis (TGA)

[0076] The TGA spectrum of the crystal form was collected using a METTLER TOLEDO thermogravimetric analyzer. The test temperature range was 35°C to 500°C, and the heating rate was 10°C / min.

[0077] (4) Dynamic moisture sorption (DVS)

[0078] The DVS spectrum of the crystal form was tested using DVS Intrinsic (SMS), the test temperature was 25°C, and the detection was performed in DMDT mode. Example

[0079] Step 1: Synthesis of methyl 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylate

[0080] 3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxylic acid (837 mg, 2.35 mmol), DMF (30 mL), NMI (1.17 g, 11.4 mmol) and methyl 4-aminopicolinate (550 mg, 3.64 mmol), TCFH (2.08 mg, 7.2 mmol) were added under ice bath, and the mixture was naturally restored to room temperature and stirred for 1 h. Water (20 mL) was added to quench the reaction, and ethyl acetate (30 mL×3) was used for extraction. The organic phases were combined, washed with water and saturated brine respectively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by Pre-HPLC to obtain the target compound (910 mg), MS: m / z =491.1, [M+H] + .

[0081] Step 2: Synthesis of 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide

[0082] The product of step 1 (910 mg) and 2 M ammonia methanol solution (15 mL) were heated to 60 °C and stirred. The system was cooled to room temperature and concentrated under reduced pressure. The crude product was separated by Pre-HPLC to obtain the target compound (711 mg). MS: m / z = 476.3, [M+H] + , 1 H NMR (400 MHz, DMSO) δ 10.81 (s, 1H), 8.46 (d, J = 5.5 Hz, 1H), 8.15(d, J = 1.7 Hz, 1H), 8.03 (s, 1H), 7.67 (dd, J = 5.5, 2.0 Hz, 1H), 7.60 (s, 1H),7.28 – 7.09 (m, 2H), 4.61 (d, J = 10.6 Hz, 1H), 4.39-7.18 (m, 1H), 3.97 (d, J =1.5 Hz, 3H), 2.47 - 2.13 (m, 2H), 1.74 (s, 3H).

[0083] Step 3: Preparation of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide

[0084] The product of step 2 was separated by the following chiral separation method: chromatographic column AD-H, column temperature 30°C, mobile phase (n-hexane-anhydrous ethanol-isopropanol-diethylamine = 80:16:4:0.05 (volume ratio)), flow rate 1 mL / min, and the component with a retention time of about 5.3 min was taken and dried.

[0085] To 5 g of the product obtained according to the above method, add 25 ml of methanol, heat and stir to reflux temperature to dissolve to obtain a clear solution, then cool to 50°C, and add 100 mL of purified water dropwise to the clear solution; after the addition is completed, cool to room temperature to grow crystals, then filter, and then vacuum dry at 45°C and -0.07 MPa to obtain 4.05 g of the target compound.

[0086] The XRPD pattern of the obtained compound is as follows: Figure 1 As shown, the obtained compound is Compound 1 Form A.

[0087] Experimental Example 1: Manual patch clamp technique to monitor the effect of the test substance on the current of stably overexpressed Nav1.8 channels

[0088] Experimental methods:

[0089] 1. Reagent preparation

[0090] Test compounds were dissolved in dimethyl sulfoxide (DMSO).

[0091] The extracellular solution was: 140 mM NaCl, 3.5 mM KCl, 1 mM MgCl2•6H2O, 2 mM CaCl2•2H2O, 10 mM D-Glucose, 10 mM HEPES, 1.25 mM NaH2PO4•2H2O, pH = 7.4 adjusted by NaOH. The intracellular solution was: 50 mM CsCl, 10 mM NaCl, 10 mM HEPES, 60 mM CsF, 20 mM EGTA, pH = 7.2 adjusted by CsOH.

[0092] 2 Experimental Materials and Instruments

[0093] 1) Patch clamp amplifier: EPC 10 (HEKA)

[0094] 2) Micromanipulator: MP225 (Sutter Instrument)

[0095] 3) Inverted microscope: MF53 (Mshot)

[0096] 4) Microelectrode pulling instrument: P97 (Sutter Instrument)

[0097] 5) Capillary glass tube: BF150-86-10 (Sutter Instrument)

[0098] 3 Experimental procedures

[0099] 1) After the compounds are prepared into solutions of specified concentrations, they are added into the drug delivery system tubing in sequence and marked.

[0100] 2) Place the cell slide in the recording chamber, select appropriate cells under an inverted microscope and adjust the position of the dosing head.

[0101] 3) Use a microelectrode puller to pull the capillary glass tube into a suitable recording electrode, then install the electrode filled with intracellular fluid into the microelectrode holder. Under an inverted microscope, adjust the micromanipulator to make the recording electrode contact the cell, and apply negative pressure to the electrode to form a high-resistance seal. At this time, perform fast capacitance compensation, and then continue to apply negative pressure to break the cell membrane and form a whole-cell recording mode. Finally, perform slow capacitance compensation and record relevant parameters.

[0102] 4) After the cell current stabilizes, drug administration begins. Each drug concentration is allowed to act for five minutes or the current is stabilized before monitoring the next concentration. The drug solution flows through the recording bath from low concentration to high concentration in sequence by gravity administration to act on the cells. A peristaltic pump is used for liquid replacement during recording.

[0103] 4 Test voltage procedure (quiescent state) and results

[0104] After the whole-cell seal is formed, the cell voltage is clamped at -120 mV, and then depolarized to 0 mV with a 50-ms square wave pulse to obtain the Nav1.8 current. This procedure is repeated every 20 s, and the maximum current induced by the square wave is monitored. After it stabilizes, the test compound is given. When the reaction stabilizes, the intensity of the current block is calculated.

[0105] After testing, the IC of the compound in Example 1 50 ≤0.06 nM.

[0106] Experimental Example 2: Study on the efficacy of compounds in the incisional pain model of SD rats / ICR mice

[0107] Experimental animals: SD rats, male, 180-220 g; ICR mice, male, 28-35 g.

[0108] Compound preparation: First, add 5% DMSO to the final volume to dissolve the test compound, and then add 10% Cremophor EL and 85% saline to the final volume.

[0109] 1. Model building

[0110] The rat / mouse was placed in an anesthesia induction box and given 3%-4% isoflurane to induce anesthesia. After anesthesia induction, a breathing mask was connected and the isoflurane concentration was adjusted to 1%-2% to prevent the rat / mouse from suffering during the operation. The rat / mouse was placed in a supine position and the surgical area was disinfected with alcohol and iodine. Under sterile conditions, a 0.5 cm longitudinal incision was made with a blade toward the finger end at 0.2 cm from the heel of the left hind foot surface, the skin and fascia were cut, the plantar muscle was separated, slightly lifted, and longitudinally cut, and then the skin was sutured with sutures and disinfected.

[0111] 2. Drug administration test

[0112] A cilium stimulation needle (Von Frey) test was performed before surgery. On the second day after the animal model was established, the animals that met the baseline requirements were selected for inclusion in the group and randomly divided into 6 groups according to the 50% PWT value, with 10 animals in each group. The double-blind test was performed, and the 50% PWT of the animals was tested before administration (baseline), 0.5 h, 2 h, 4 h or 6 h after administration, respectively. The single administration was performed. The model control group was given normal saline, and the compound group was given the compound according to the dose in the following table.

[0113] Table 1: PWT 50% values ​​of each group of compound mice at different time points (g, Mean±SEM, n=10)

[0114] Group Preoperative Before postoperative medication 0.5 h 2 h 4 h 6 h Model comparison 1.2±0.15 0.22±0.03 0.2±0.04 0.21±0.02 0.24±0.06 0.26±0.08 Example 1 Compound 20 mg / kg 1.29±0.28 0.22±0.03 0.52±0.31 0.72±0.23 0.53±0.26 0.4±0.18

[0115] Table 2: PWT 50% values ​​of each group of compound rats at different time points (g, Mean±SEM, n=10)

[0116] Group Preoperative Before postoperative medication 0.5 h 2 h 4 h 6 h Model comparison 14.74±1.8 4±1.19 4.7±1.06 3.87±1.41 4.49±1.86 4.71±1.21 Example 1 Compound 20 mg / kg 15.41±3.46 3.92±1.16 6.06±2.41 8.04±2.15 5.85±2.25 5.31±0.99

[0117] In the incisional pain models of mice and rats, the compound of Example 1 had good analgesic activity and a long analgesic duration at a dose of 20 mg / kg.

[0118] Experimental Example 3: Study on the efficacy of compounds in the rat spinal nerve ligation model (SNL)

[0119] Experimental animals: SD rats, male, 180-220 g.

[0120] Compound preparation: First, add 5% DMSO to the final volume to dissolve the test compound, and then add 10% Cremophor EL and 85% saline to the final volume.

[0121] 1. Model building

[0122] SD rats were anesthetized with isoflurane breathing. The SD rats were placed in a prone position and the surgical area was shaved locally. The surgical area was disinfected with alcohol and iodine. A longitudinal 3-4 cm incision was made at the level of the lower lumbar / upper sacrum under sterile conditions to expose the left paraspinal muscles. The paraspinal muscles were bluntly separated with small blunt scissors and the L6 transverse process was removed. The L4 and L5 spinal nerves were separated and exposed. The L5 spinal nerve was ligated with 6-0 silk thread. Ceftriaxone was used to disinfect the wound to prevent infection. The muscles and skin were sutured layer by layer and disinfected with iodine. After the operation, the animals were placed on a warm electric blanket for warmth. After the animals were fully awake, they were returned to the cage for feeding and their behavior was observed after the operation.

[0123] 2. Drug administration test

[0124] A Von Frey test was performed before surgery, and leg-lifting training was performed on the 5th and 6th days after surgery. Animals with a baseline of 2-5 g were selected and divided into groups according to the 50% PWT value, with 10 animals in each group. A single dose was administered in a blind manner on the second day, and the 50% PWT of the animals was tested before (Baseline), 2 h, 4 h, 6 h or 8 h after administration. The model control group was given normal saline, and the compound group was given the compound according to the dose in the following table.

[0125] Table 3: PTW 50% values ​​of each group of compound rats at different time points (g, Mean±SEM, n=10)

[0126] Group Preoperative Before postoperative medication 2 h 4 h 6 h 8 h <![CDATA[AUC 0-8h ]]> Model comparison 14.71±2.89 2.69±1.15 2.92±1.3 2.83±1.36 2.5±1.15 2.53±1.34 21.81±3.96 Example 1 Compound 20 mg / kg 14.33±3.97 2.69±1.04 5.1±2.28 5.57±2.25 3.67±1.41 3.4±1.11 34.28±7.86

[0127] In the rat spinal nerve ligation model, the compound of Example 1 had good analgesic activity and a longer analgesic duration at a dose of 20 mg / kg.

[0128] Experimental Example 4: Study on the efficacy of compounds in CFA-induced inflammatory pain model in rats

[0129] Experimental animals: SD rats, male, 180-220 g.

[0130] Compound preparation: First, add 5% DMSO to the final volume to dissolve the test compound, and then add 10% Cremophor EL and 85% saline to the final volume.

[0131] 1. Model building

[0132] SD rats were anesthetized with isoflurane, the right hind foot was disinfected, the needle was inserted subcutaneously from the posterior half of the sole, 50 μL of complete Freund's adjuvant (CFA, 1 mg / mL) was injected subcutaneously in the middle of the sole, and the needle tip was slowly rotated to avoid leakage. After modeling, local redness and swelling of the sole, difficulty walking, and retraction of the affected foot were observed.

[0133] 2. Drug administration test

[0134] A cilium stimulation needle (Von Frey) test was performed before surgery. On the second day after animal modeling, animals that met the baseline requirements were selected and randomly divided into 6 groups according to the 50% PWT value, with 10 animals in each group. Double-blind test administration was performed, and the 50% PWT of the animals was tested before administration (baseline), 0.5 h, 2 h, and 8 h after administration. The model control group was given normal saline, and the compound group was given the compound according to the dose in the following table.

[0135] Table 4: PWT 50% values ​​of rats in each group of the compounds of the present invention at different time points (g, Mean ± SEM, n = 10)

[0136] Group Preoperative Before postoperative medication 0.5 h 2 h 8 h <![CDATA[AUC 0-8h ]]> Model comparison 13.44±3.71 3.13±1.2 3.32±1.39 3.21±1.25 3.11±1.19 25.08±5.52 Example 1 Compound 50 mg / kg 13.16±2.19 3.1±1.14 4.78±2.67 6.15±3.75 3.87±1.42 36.16±11.08

[0137] The compound of Example 1 exhibited good analgesic activity in the CFA-induced inflammatory pain model in rats.

[0138] Experimental Example 5: Study on the efficacy of compounds in the acetic acid writhing model in mice

[0139] Experimental animals: ICR mice, male, 20-24 g.

[0140] Compound preparation: First, add 5% DMSO to the final volume to dissolve the test compound, and then add 10% Cremophor EL and 85% saline to the final volume.

[0141] 1. Model establishment The rats were randomly divided into groups according to their body weight, with 10 rats in each group. 0.8% (v / v) acetic acid solution was injected intraperitoneally with an injection volume of 10 mL / kg.

[0142] 2. Drug administration test

[0143] The drug was administered 60 min before modeling. After the injection of acetic acid solution, the number of writhing of mice was observed. The behavior of the mice with abdominal concavity, trunk and hind limb extension, and buttocks raised was called a complete writhing reaction. The number of writhing of mice within 0-15 min and 15-30 min was observed and recorded for a total of 30 min. Inhibition rate = (number of writhing in the control group - number of writhing in the drug administration group) / number of writhing in the control group × 100%. The model control group was given physiological saline, and the other groups were given compounds according to the dosage in the table below.

[0144] Table 5: Number of writhing reactions of animals in each group of compound mice (Mean±SD, n=10)

[0145] Group Dosage (mg / kg) Number of twists Inhibition rate (%) Model control group / 49 ± 16 - Example 1 Compound 20mg / kg 30±14 38.35

[0146] The compound of Example 1 of the present invention can well inhibit the writhing effect at a low dose of 20 mg / kg.

[0147] Experimental Example 6 Stability Test

[0148] The compound prepared in Example 1 of the present invention was subjected to high temperature (60°C ± 2°C), high humidity (45°C ± 2°C / 75%RH ± 5%RH), light (4500 lx ± 500 lx and ≥ 0.80×10 2 μW / cm 2), with package lighting (4500 lx±500 lx and ≥0.80×10 2 μW / cm 2 ) conditions for stability testing.

[0149] The results showed that the compounds of the present invention had excellent chemical stability and crystal stability under light, packaged light, high temperature, high humidity and light-shade conditions. The purity on 29 days was basically unchanged from the purity on 0 days, no crystal form change occurred, and the HPLC purity was greater than 99.5%.

[0150] Experimental Example 7 Solubility Test

[0151] The solid form of the compound prepared in Example 1 of the present invention was tested for solubility in different solutions, and the test results are as follows: the solubility of compound 1 in water and a series of pH buffer solutions in the physiological pH range is about 0.01~5.7μg / ml. The solubility of compound 1 crystal form A in FeSSIF (simulated full intestinal fluid) and FaSSIF (simulated hungry intestinal fluid) is >55μg / mL and ≥14μg / mL, respectively. This shows that compound 1 is a poorly soluble compound, and the crystal form A of the present invention can improve its solubility.

Claims

1. A method for preparing the crystalline form A of 4-((2R,3S,5R)-3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxamide, characterized in that: The XRPD pattern of Form A includes o , 13.2±0.2 o , 14.5±0.2 o , 16.0±0.2 o , 17.8±0.2 o , 18.6±0.2 o , 19.3±0.2 o , 19.6±0.2 o , 20.6±0.2 o , 21.4 ±0.2 o , 21.9 ±0.2 o , 23.6±0.2 o , 24.3±0.2 o , 25.0±0.2 o , 25.6±0.2 o , 26.0±0.2 o , 27.0±0.2 o , 27.9±0.2 o , 28.5±0.2 o , 29.3±0.2 o , 36.3±0.2 o Characteristic peak at the diffraction angle (2θ) at ; The preparation method of the crystal form A comprises the following steps: mixing the chiral separation product of the compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylic acid methyl ester with methanol, heating the mixture to dissolve, then cooling the mixture to 50-60° C., adding water, and cooling the mixture to 0-30° C.; The chromatographic column for chiral separation is AD-H, and the mobile phase for chiral separation is a mixed solution of n-hexane-anhydrous ethanol-isopropanol-diethylamine in a volume ratio of (70-90): (10-20): (1-10): (0.01-1); The mass volume ratio of the product obtained by chiral separation of the compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylic acid methyl ester to methanol, in g / ml, is 1:2-30; The mass volume ratio of the product obtained by chiral separation of the compound 4-(3-(3,4-difluoro-2-methoxyphenyl)-5-methyl-5-(trifluoromethyl)thiolane-2-carboxamido)pyridine-2-carboxylic acid methyl ester to water is 1:5-30 in g / ml.

2. The method for preparing the crystal form A according to claim 1, characterized in that: The diffraction angle (2θ) in the XRPD spectrum of the crystalline form A is 14.5°±0.2 o The diffraction peak intensity is 100%, and the diffraction angle (2θ) is 12.8°±0.2 o The diffraction peak intensity is ≥70%.

3. The method for preparing the crystalline form A according to claim 1 or 2, characterized in that: The XRPD pattern of Form A includes peaks at diffraction angles (2θ) substantially the same as those shown in FIG. 1 .

4. The method for preparing the crystal form A according to claim 1 or 2, characterized in that: The XRPD pattern of the crystalline form A is shown in FIG1 .

5. The method for preparing the crystal form A according to claim 1, characterized in that: The mobile phase for chiral separation is a mixed solution of n-hexane-anhydrous ethanol-isopropanol-diethylamine in a volume ratio of 80:16:4:0.05.

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